Process of making spore-containing cultures of japanese beetle milky disease bacteria



United States Patent 3,308,038 PROQESS OF MAKING SPORE-CONTAININGCULTURES 0F JAPANESE BEETLE MlLKY DISEASE BACTERIA Robert A. Rhodes,George R. Hrubant, and Margaret S. Roth, Peoria, Ill., assiguors to theUnited States of America as represented by the Secretary of AgricultureNo Drawing. Filed Nov. 13, 1964, Ser. No. 411,161 3 Claims. (Cl. 195-96)A nonexclusive, irrevocable, royalty-free license in the inventionherein described, throughout the world for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention relates to a commercially promising in vitro process forreproducibly obtaining appreciable extents of sporulated cell formationin agar plate colonies of Bacillus popilliae that have been plated fromvegetative cultures of the organism, the in vitro-generated spores beingnot only apparent-1y capable of producing the highly fatal milky diseasewhen injected into Japanese beetle larvae but also being capable ofproducing vegetative cultures, injections of which are highly pathogenicfor the larvae of the Japanese beetle, Popillia japonica Newman, therebysimplifying the reproducible provision of Bacillus popilliae cellsneeded for sporogenesis studies that may lead to extensive commercial invitro production of viable, highly infective spores for the biologicalcontrol of the Japanese beetle in place of the scarce and costlyspore-containing powdered tissue obtained upon the death of laboriouslyinjected Japanese "beetle larvae.

Nearly three decades ago Dutky discovered that the highly lethal milkydisease of Japanese beetles is caused therein by ingestion of infectivespores of two closely related bacteria, namely Bacillus popilliae andBacillus lentimorbus. Dutky also conceived and developed the onlypresently available biological control agent by injecting hand collectedJapanese beetle larvae with viable spores of the above pathogens,allowing the larvae to die from the resulting growth of billions ofbacterial cells therein, then dessicating the spore-containing deadlarvae, and grinding their tissue to a dispersable infective powder.

Despite extensive research by many qualified rnicro-' biologists, it hasheretofore been impossible to reproducibly obtain any in vitro formationof sporulated rather than exclusively vegetative Bacillus popilliaecells, thereby greatly hampering in vitro efforts to study thesporogenesis of the milky disease bacteria which, sporulation in natureoccurs only in the body of the infected Japanese beetle larvae where,following a prolonged latency, sporulation occurs very explosivelyshortly before the death of the host. While the maintenance 'of milkydisease bacterial spores in dried films of infected Popillia japonicahemolymph provides a water dispersible material that is infective wheninjected directly into the hemocoeles of Japanese beetle larvae, suchprior art materials do not represent pure cultures in themicrobiological sense, are not suitable as stock inocula for readilyproducing vegetative cultures of the milky disease microorganisms, anddo not provide a means for studying the very complex sporogenesis of themilky disease bacteria that may lead to large scale in vitro sporulationand less costly products for the biological control of the detestedJapanese beetle.

A major object of our invention is an in vitro microbiological processfor obtaining pure cultures of Japanese beetle milky disease bacteriathat consistently comprise an appreciable proportion of viable andinfective sporulated cells.

Another object is an in vitro process for reproducibly culturing storageand heat-resistant spore-containing colonies of certain strains of milkydisease bacteria, which 3,308,038 Patented Mar. 7, 1967 spore-containingmaterial can be employed both as an inexpensive and effective inoculumfor bulk scale fermentations of infective vegetative cells of the milkydisease bacteria and as a dependable source of a viable sporecontainingmaterial that may permit a widespread biological control program.

Other and related objects will become apparent the following detaileddisclosure and claims.

In accordance with the broad objects of our invention we have nowdiscovered an in vitro technique for reproducibly obtaining limited buthighly useful extents of sporulation (about 3 percent to as high asabout 5 percent of the total cell population being in the spore form) ofcertain pure strains of Japanese milky disease organisms, which viablebacterial spores are resistant to heat and to dehydration, do notrequire frequent serial transfers to preserve viability as do thecorresponding vegetative cell, and that constitute a convenient inoculumfor the extensive propagation of the pathogenic vegetative form of theorganism in liquid media. Thus, it has now been found that appreciableand useful extents of sporulated Bacillus popilliae are reproduciblyformed when the lyophilized vegetative cells of certain specificsubstrains of Bacillus popilliae, i.e., NRRL B-2309L and NRRL B-2309Sthat we have isolated from plate cultures of the known wild type strainNRRL B2309 are first cultured for 18-24 hours in shaken flasks or withaeration at the rate of at least 0.15 v./v./min. and up to 0.5v./v./min. in an aqueous medium critically containing as separatelyautoclaved constituents 0.2 percent of a fermentable carbohydrate suchas glucose, fructose, or trehalose, 1.5-2.0 percent of commerciallyavailable unfractionated yeast extract, and suflicient K HPO buffer toinitially provide a pH of 7.2 to 7.5 (0.3% provides a pH of 7.3), thensparingly inoculating the surface of carbohydrate-free acetate-agarplates with 0.3 ml. of a 10 dilution (using 0.1% tryptone as diluent) ofthe exclusively vegetative cell culture, said plates having beenprepared by adding to an autoclaved solution containing 2.0 percent ofprewashed agar and 1.5 to 2.0 percent of yeast extract a separatelyautoclaved mixture containing 0.035 to 0.070 percent (w./v.) of sodiumacetate and 0.3 percent of K HPO likewise based on the solution, whichthusly formed carbohydrate-free acetate-agar medium avoids the formationof an inhibitor substance, at most within several hours afterinoculating the plates, overlaying the same with 5 ml. of an autoclaved1 percent solution of granular agar that was first exhaustively washedin 10-12 changes of deionized water, incubating the overlayed inoculatedplates at 30 C. for about 42 days to obtain colonial growth, at whichtime microscopic counts show that only when the plates have beensparingly inoculated as to provide not more than about 30 uncrowdedcolonies per plate, proportions amounting to about 3 percent to about 5percent of the total bacterial cell count of the otherwise exclusivelyvegetative colonies are in the spore form. Suspensions of thespore-containing material, which may be given even greater ability togerminate by subjection to thenmal shock at 50 C. for 15 minutes, maycause milky disease when sufficient viable cells are injected intoJapanese beetle larvae, and can be employed as an available inoculum forthe growth of pathogenic vegetative cultures.

The following particulars will contribute to a better understanding andemployment of the above technique.

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Contrary to Dutkys teaching in Steinhaus (Ed.) Insect Pathology,- vol.2, pp. l15, Academic Press (1963) to the effect that Bacillus popilliaeorganisms are anaerobic, we have found that substrains NRRL B2309L andNRRL B-2309S require oxygen and that their rate of growth in liquidculture is directly proportional to the supply of oxygen-containing air,the minimum required aeration being equivalent'to 0.15 v./v./min.Aerations exceeding 0.5 v./v./rnin. do not further increase the rate orextent of growth, and are particularly undesirable since in aqueousmedia increased rates and extents of vegetative proliferation result indiminished viability and necessitate more frequent serial transfers toprevent loss of the organism, which ordinarily promptly loses viabilityon reaching a late log phase population of about 5 10 viable vegetativecells per ml.

EXAMPLE Lyophilized vegetative cells of isolated strains of Bacilluspopilliae NRRL B-230'9L and Bacillus popilliae NRRL B-2309S werecultured in respective 5 ml. Erlenmeyer flasks each containing 100 ml.of a distilled water basal medium containing 1.5 percent of separatelyautoclaved yeast extract obtained from an established supplier ofmicrobiological media, 0.3 percent K HPO and 0.2 percent glucose. Thesterilized triplicate flasks for each strain were incubated at 30 C. ona rotary shaker until the late log phase cell populations reached avalue of about 5 vegetative cells per ml. (16-24 hours depending on therate of agitation, i.e., aeration). Then without substantial delay(serial transfer and reculture otherwise being required), 0.3 ml.portions of the respective vegetative cultures that had just beendiluted 10- million fold (10 with a 0.1 percent tryptone solution werespread on triplicate acetate-agar plates in which the medium had beenprepared by adding to a distilled water solution containing 1.5 percentof the said separately autoclaved yeast extract and 0.1 percent K HPO2.0 percent of deionized water-washed agar, and 0.070 percent (W./v.) ofCH COONa before sterilizing. The acetate agar plates (35 ml. beforesolidification) had a pH of 7.2-7.4. The seeded plates were thenpromptly overlayed with 5 ml. of a 1 percent solution of washed agar andincubated for 42 days at 30 C., during which period the colonial growththereon was periodically examined for the typical refractile spores andparaspores by phase microscopy and in stained preparations. Significantsporulation did not occur until the last few days of the 6-week period,but pre-sporulation swellings were observed considerably sooner.Sporulation of Bacillus popilliae NRRL B-2309L and Bacillus popilliaeNRRL B2309S occurs only if the vegetative colonies are widely spaced andthere are not more than about 30 colonies per plate, the latter beingcontrolled by proper dilutions of the liquid culture population per ml.;under the described conditions about 3 percent of the colonial B-2309Scells and up to 5 percent of the B2309L cells are found to have becomespores after 6 weeks incubation.

Appreciable spore formation occurred only in the presence of a properamount of added sodium acetate, concentrations above 17 mM. beingsomewhat inhibitory. Colony formation, however, was not dependent on thepresence or absence of acetate in the agar medium. Similarly, colonyformation is not influenced by the presence or absence of fermentablecarbohydrate, but spore formation is severely restricted by even smallamounts of glucose in the acetate agar. Trehalose is considerably lessinhibitory to sporulation by Bacillus popilliae than is glucose.Bacto-agar contents outside the range of 1.5-2.5 percent in the originalsolid medium layer markedly reduced the extent of sporulation, as didthe substitution of other agars. Likewise, increasing the yeast extractconcentration from the operative level of 1.5 percent to a concentrationof 2.0 percent completely prevented spore formation while lowering theyeast extract concentration to 1.0 percent distinctly depressedsporulation. Substitutions of yeast extract fractions were of little orno value, and the whole or partial substitution of other organic sourcesof nitrogen for yeast extract in the acetate agar invariably restrictedeither growth or sporulation.

Subjecting suspensions of the obtained exemplary sporecontaining colonymaterials to a temperature of 50 C. for 15 minutes expectedly killed thevegetative cells but improved the germinability of the spores.Vegetative cells of liquid cultures established from the germinatedspores were shown to be pathogenic for milky disease when injected intohealthy Japanese beetle larvae. Having fully disclosed our invention weclaim: 1. An in vitro process for inducing sporulation to the extent ofabout 3 percent to about 5 percent based on the total number of Japanesebeetle milky disease bacterial cells present in the form of discretecolonies grown on a solid culture medium, said process comprisinginoculating a distilled water medium containing 1.5 percent by weight ofseparately autoclaved yeast extract, 0.3 percent dipotassium phosphatebuffer, and 0.2 percent of a fermentable carbohydrate selected from thegroup consisting of tre-halose, glucose, and fructose with viablevegetative cells obtained by liquid culture of a Japanese beetle milkydisease organism selected from the group consisting of Bacilluspopilliae NRRL 13-23091. and Bacillus popilliae NRRL B-2309S,

fermenting the inoculated medium at 30 C. in the presence of aerationequivalent to 0.l50.5 v./ v./ min. until the resulting vegetative cellsreach a late log phase population of about 5x10 cells per ml.,

promptly diluting a small amount of the fermented liquid medium with 10"volumes of a 0.1-percent tryptone solution, spreading 0.3 ml. aliquotsof the diluted fermentation medium on the surface of each of replicatedsolid media plates prepared by adding as separately autoclavedcomponents 0.10 percent to 0.150 percent by weight of K H PO based onthe aqueous solution and 0.035 percent to 0.070 percent (w./v.) ofsodium acetate to a distilled water solution containing 1.5 percent ofyeast extract and 2.0 percent of agar,

promptly overlaying the thusly inoculated plates with 5 ml. of anautoclaved l-percent solution of exhaustively washed agar, incubatingthe overlaid plates at 30 C. for 42 days whereby to obtain not more thanabout 30' colonies per plate in which colonies about 3 percent to about5 percent of the individual cells thereof are in the spore form, v

subjecting suspensions of the above colonial material to heating for 15minutes at 50 C. to kill the vegetative cells and provide an agar platehaving thereon only sporulated cells of a said Japanese beetle milkydisease organism, said sporulated cells having improved germinability.

2. The process of claim 1 wherein the milky disease organism is Bacillusp opilliae NRRL B-2309S, the fermentable carbohydrate is glucose, andthe aeration rate is 0.50 v./v./min.

3. The process of claim 1 wherein the milky disease organism is Bacilluspopilliae NRRL B2309L, the fermentable carbohydrate is glucose, and theaeration rate is 0.50 v./v./min.

No references cited.

A. LOUIS MONACELL, Primary Examiner.

L. M. SHAPIRO, Assistant Examiner,

1. AN IN VITRO PROCESS FOR INDUCING A SPORULATION TO THE EXTENT OF ABOUT3 PERCENT TO ABOUT 5 PERCENT BASED ON THE TOTAL NUMBER OF JAPANESEBEETLE MILKY DISEASE BACTERICAL CELLS PRESENT IN THE FORM OF DISCRETECOLONIES GROWN ON A SOLID CULTURE MEDIUM, SAID PROCESS COMPRISINGINOCULATING A DISTILLED WATER MEDIUM CONTAINING 1.5 PERCENT BY WEIGHT OFSEPARATELY AUTOCLAVED YEAST EXTRACT, 0.3 PERCENT DIPOTASSIUM PHOSPHATEBUFFER, AND 0.2 PERCENT OF A FERMENTABLE CARBOHYDRATE SELECTED FROM THEGROUP CONSISTING OF TREHALOSE, GLUCOSE, AND FRUTOSE WITH VIABLEVEGETATIVE CELLS OBTAINED BY LIQUID CULTURE OF A JAPANESE BEETLE MILKYDISEASE ORGANISM SELECTED FROM THE GROUP CONSISTING OF BACILLUSPOPILLIAE NRRL B-2309L AND BACILLUS POPILLIAE NRRL B-2309S, FERMENTINGTHE INOCULATED MEDIUM AT 30*C. IN THE PRESENCE OF AERATION EQUIVALENT TO0.15-0.5 V./V./MIN. UNTIL THE RESULTING VEGETATIVE CELLS REACH A LATELOG PHASE POPULATION OF ABOUT 5X10**8 CELLS PER ML., PROMPTLY DILUTING ASMALL AMOUNT OF THE FERMENTED LIQUID MEDIUM WITH 10**7 VOLUMES OF A0.1-PERCENT TRYPTONE SOLUTION, SPREADING 0.3 ML. ALIQUOTS OF THE DILUTEDFERMENTATION MEDIUM ON THE SURFACE OF EACH OF REPLICATED SOLID MEDIAPLATES PREPARED BY ADDING AS SEPARATELY AUTOCLAVED COMPONENTS 0.10PERCENT TO 0.150 PERCENT BY WEIGHT OF K2HPO4 BASED ON THE AQUEOUSSOLUTION AND 0.035 PERCENT TO 0.070 PERCENT (W./V.) OF SODIUM ACETATE TOA DISTILLED WATER SOLUTION CONTAINING 1.5 PERCENT OF YEAST EXTRACT AND2.0 PERCENT OF AGAR, PROMPTLY OVERLAYING THE THUSLY INOCULATED PLATESWITH 5 ML. OF AN AUTOCLAVED 1-PERCENT SOLUTION OF EXHAUSTIVELY WASHEDAGAR, INCUBATING THE OVERLAID PLATES AT 30*C. FOR 42 DAYS WHEREBY TOOBTAIN NOT MORE THAN ABOUT 30 COLONIES PER PLATE IN WHICH COLONIES ABOUT3 PERCENT TO ABOUT 5 PERCENT OF THE INDIVIDUAL CELLS THEREOF ARE IN THESPORE FORM, SUBJECTING SUSPENSION OF THE ABOVE COLONIAL MATERIAL TOHEATING FOR 15 MINUTES AT 50* C. TO KILL THE VEGETATIVE CELLS ANDPROVIDE AN AGAR PLATE HAVING THEREON ONLY SPORULATED CELLS OF A SAIDJAPANESE BEETLE MILKY DISEASE ORGANISM, SAID SPORULATED CELLS HAVINGIMPROVED GERMINABILITY.